RibH

ABSTRACT

The invention provides ribH polypeptides and polynucleotides encoding ribH polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ribH polypeptides to screen for antibacterial compounds.

This application is a divisional of U.S. application Ser. No.08/977,553, filed Nov. 25, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to novel polynucleotides and polypeptides of the lumazinesynthase (β-subunit) family, hereinafter referred to as “ribH”.

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

Riboflavin (vitamin B2) is a member of the B complex of vitamins whichfunction as coenzymes in metabolic reactions. Riboflavin has twocoenzyme forms, flavin mononucleotide (FMN) and flavin adeninedinucleotide (FAD) which act in oxidation-reduction reactions such asthe cytochrome system of electron transport and the oxidativedegradation of pyruvate, fatty acids and amino acids. The firstcommitted step in the biosynthesis of riboflavin is the opening of theimidazole ring of GTP. In the presence of 3 H₂O and Mg⁺⁺, the C-8 of GTPis released as formate accompanied by the release of pyrophosphate bythe action of GTP cyclohyrolase II (GCH2; EC 3.5.4.25). This enzymefunction is encoded by ribA in bacteria and rib1 in yeast species.Through a series of steps, involving 3,4-dihydroxy-2-butanone 4phosphate synthase (ribA), 6,7-dimethyl-8-ribityllumazine synthase(ribH), riboflavin synthase (ribB), pyrimidine deaminase and pyrimidinereductase (ribG), enzymes encoded by genes within the riboflavinbiosynthesis operon, riboflavin is formed. Because the genes requiredfor riboflavin biosynthesis are present in many pathogenicmicroorganisms, and since riboflavin biosynthesis has shown to berequired for virulence in the swine pathogen Actinobacilluspleuropneumoniae (Fuller, T E, et al. (1996) A riboflavin auxotroph ofActinobacillus pleuropneumoniae is attenuated in swine. Infect. Immun.64:4659-4664), these gene products represent broad spectrumantibacterial as well as antifungal targets.

The frequency of Staphylococcus aureus infections has risen dramaticallyin the past few decades. This has been attributed to the emergence ofmultiply antibiotic resistant strains and an increasing population ofpeople with weakened immune systems. It is no longer uncommon to isolateStaphylococcus aureus strains which are resistant to some or all of thestandard antibiotics. This phenomenon has created a demand for both newanti-microbial agents, vaccines, and diagnostic tests for this organism.

Clearly, there exists a need for factors, such as the ribH embodimentsof the invention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists to find ways to prevent,ameliorate or correct such infection, dysfunction and disease.

Certain of the polypeptides of the invention possess amino acid sequencehomology to a known Bacillus amyloliquefaciens ribH protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel ribH polypeptides by homology between the amino acidsequence set out in Table 1 [SEQ ID NO: 2] and a known amino acidsequence or sequences of other proteins such as Bacillusamyloliquefaciens ribH protein.

It is a further object of the invention to provide polynucleotides thatencode ribH polypeptides, particularly polynucleotides that encode thepolypeptide herein designated ribH.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding ribH polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO:1] which includes a full lengthgene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel ribH protein from Staphylococcus aureus comprising the amino acidsequence of Table 1 [SEQ ID NO:2], or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the as Staphylococcus aureus WCUH29 strain, which nucleic acid iscontained in the deposited strain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding ribH, particularly Staphylococcus aureus ribH,including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of ribH and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStaphylococcus aureus referred to herein as ribH as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of ribH polypeptide encoded by naturally occurring alleles ofthe ribH gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned ribH polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing ribHexpression, treating disease, assaying genetic variation, andadministering a ribH polypeptide or polynucleotide to an organism toraise an immunological response against a bacteria, especially aStaphylococcus aureus bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to ribH polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention there are providedantibodies against ribH polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided ribH agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a ribH polynucleotide or a ribH polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to novel ribH polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel ribH of Staphylococcusaureus, which is related by amino acid sequence homology to Bacillusamyloliquefaciens ribH polypeptide. The invention relates especially toribH having the nucleotide and amino acid sequences set out in Table 1as SEQ ID NO: 1 and SEQ ID NO: 2 respectively ribH.

TABLE 1 ribH Polynucleotide and Polypeptide Sequences (A) Sequences fromStaphylococcus aureus ribH polynucleotide sequence [SEQ ID NO:1]. 5′-ATGAATTTTGAAGGTAAATTAATTGGAAAAGATTTGAAAGTTGCAATCGTAGTTAGTCGATTTAATGATTTTATCACTGGAAGATTACTTGAAGGTGCAAAAGATACTTTGATTCGACATGATGTTAATGAAGACAATATTGATGTAGCATTTGTTCCTGGTGCGTTTGAAATTCCTTTAGTAGCTAAAAAATTAGCCTCATCAGGAAATTATGATGCAATAATTACATTAGGATGCGTAATTCGCGGTGCTACGTCTCATTATGATTATGTTTGTAATGAAGTCGCGAAAGGTGTTTCTAAAGTAAATGATCAAACTAATGTACCAGTCATATTTGGTATTTTAACGACTGAAAGTATTGAACAAGCTGTGGAAAGAGCAGGTACGAAAGCTGGTAATAAAGGTGCCGAAGCAGCAGTAAGTGCAATTGAAATGCCTAATTTATTAAAATCTATAAAAGCATAG-3′ (B) Staphylococcus aureus ribHpolypeptide sequence deduced from the polynucleotide sequence in thistable [SEQ ID NO:2]. NH₂-MNFEGKLIGKDLKVAIVVSRFNDFITGRLLEGAKDTLIRHDVNEDNIDVAFVPGAFEIPLVAKKLASSGNYDAIITLGCVIRGATSHYDYVCNEVAKGVSKVNDQTNVPVIFGILTTESIEQAVERAGTKAGNKGAEAAVSAIEMANLLKSIKA-COOH

Deposited materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, andreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

The deposited strain contains the full length ribH gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

One aspect of the invention there is provided an isolated nucleic acidmolecule encoding a mature polypeptide expressible by the Staphylococcusaureus WCUH 29 strain contained in the deposited strain: Furtherprovided by the invention are ribH nucleotide sequences of the DNA inthe deposited strain and amino acid sequences encoded thereby. Alsoprovided by the invention are ribH polypeptide sequences isolated fromthe deposited strain and amino acid sequences derived therefrom.

Polypeptides

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2] (in particular the mature polypeptide) as well as polypeptidesand fragments, particularly those which have the biological activity ofribH, and also those which have at least 70% identity to a polypeptideof Table 1 [SEQ ID NO:1] or the relevant portion, preferably at least80% identity to a polypeptide of Table 1 [SEQ ID NO:2 and morepreferably at least 90% similarity (more preferably at least 90%identity) to a polypeptide of Table 1 [SEQ ID NO:2] and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to a polypeptide of Table 1 [SEQ ID NO:2] and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

The invention also includes polypeptides of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the amino terminus, X is hydrogen, and at the carboxylterminus, Y is hydrogen or a metal, R₁ and R₃ are any amino acidresidue, m is an integer between 1 and 1000 or zero, n is an integerbetween 1 and 1000 or zero, and R₂ is an amino acid sequence of theinvention, particularly an amino acid sequence selected from Table 1. Inthe formula above R₂ is oriented so that its amino terminal residue isat the left, bound to R₁, and its carboxy terminal residue is at theright, bound to R₃. Any stretch of amino acid residues denoted by eitherR group, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with ribH polypeptides fragments maybe “free-standing,” or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or ofvariants thereof, such as a continuous series of residues that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus. Degradation forms of the polypeptides of theinvention in a host cell, particularly a Staphylococcus aureus, are alsopreferred. Further preferred are fragments characterized by structuralor functional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and tum-forming regions, coil and coil-forming regions, hydrophilicregions, hydrophobic regions, alpha amphipathic regions, betaamphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of ribH, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

In addition to the standard single and triple letter representations foramino acids, the term “X” or “Xaa” may also be used in describingcertain polypeptides of the invention. “X” and “Xaa” mean that any ofthe twenty naturally occurring amino acids may appear at such adesignated position in the polypeptide sequence.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the ribH polypeptide havinga deduced amino acid sequence of Table 1 [SEQ ID NO:2] andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1], a polynucleotide of the inventionencoding ribH polypeptide may be obtained using standard cloning andscreening methods, such as those for cloning and sequencing chromosomalDNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells asstarting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such as asequence given in Table 1 [SEQ ID NO:1], typically a library of clonesof chromosomal DNA of Staphylococcus aureus WCUH 29 in E.coli or someother suitable host is probed with a radiolabeled oligonucleotide,preferably a 17-mer or longer, derived from a partial sequence. Clonescarrying DNA identical to that of the probe can then be distinguishedusing stringent conditions. By sequencing the individual clones thusidentified with sequencing primers designed from the original sequenceit is then possible to extend the sequence in both directions todetermine the full gene sequence. Conveniently, such sequencing isperformed using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).(see in particular Screening By Hybridization 1.90 and SequencingDenatured Double-Stranded DNA Templates 13.70). Illustrative of theinvention, the polynucleotide set out in Table 1 [SEQ ID NO:1] wasdiscovered in a DNA library derived from Staphylococcus aureus WCUH 29.

The DNA sequence set out in Table 1 [SEQ ID NO:1] contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 [SEQ ID NO:2] with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. The polynucleotide of SEQ ID NO: 1,between nucleotide number 1 and the stop codon which begins atnucleotide number 463 of SEQ ID NO:1, encodes the polypeptide of SEQ IDNO:2.

RibH of the invention is structurally related to other proteins of thelumazine synthase (β-subunit) family.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence in Table 1 [SEQ ID NO:1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro-protein sequence. The polynucleotidemay also contain non-coding sequences, including for example, but notlimited to non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences, termination signals, ribosome binding sites,sequences that stabilize MRNA, introns, polyadenylation signals, andadditional coding sequence which encode additional amino acids. Forexample, a marker sequence that facilitates purification of the fusedpolypeptide can be encoded. In certain embodiments of the invention, themarker sequence is a hexa-histidine peptide, as provided in the pQEvector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad.Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al., Cell 37: 767(1984). Polynucleotides of the invention also include, but are notlimited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofcomprising nucleotide 1 to the nucleotide immediately upstream of orincluding nucleotide 463 set forth in SEQ ID NO:1 of Table 1, both ofwhich encode the ribH polypeptide.

The invention also includes polynucleotidcs of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the 5′ end of the molecule, X is hydrogen, and at the 3′ endof the molecule, Y is hydrogen or a metal, R₁ and R₃ is any nucleic acidresidue, m is an integer between 1 and 3000 or zero, n is an integerbetween 1 and 3000 or zero, and R₂ is a nucleic acid sequence of theinvention, particularly a nucleic acid sequence selected from Table 1.In the polynucleotide formula above R₂ is oriented so that its 5′ endresidue is at the left, bound to R₁, and its 3′ end residue is at theright, bound to R₃. Any stretch of nucleic acid residues denoted byeither R group, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer. In apreferred embodiment m and/or n is an integer between 1 and 1000.

It is most preferred that the polynucleotides of the inventions arederived from Staphylococcus aureus, however, they may preferably beobtained from organisms of the same taxonomic genus. They may also beobtained, for example, from organisims of the same taxonomic family ororder.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus ribH havingan amino acid sequence set out in Table 1 [SEQ ID NO:2]. The term alsoencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, interruptedby integrated phage or an insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having adeduced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingribH variants, that have the amino acid sequence of ribH polypeptide ofTable 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, that do not alter the properties and activitiesof ribH.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding ribH polypeptide having an amino acid sequence set out in Table1 [SEQ ID NO:2], and polynucleotides that are complementary to suchpolynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding ribH polypeptide andpolynucleotides complementary thereto. In this regard, polynucleotidesat least 90% identical over their entire length to the same areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at about 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof, and isolating said DNA sequence. Fragments usefulfor obtaining such a polynucleotide include, for example, probes andprimers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding ribH and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the ribH gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the ribH gene may be isolated byscreening using a DNA sequence provided in Table 1 [SEQ ID NO: 1] tosynthesize an oligonucleotide probe. A labeled oligonucleotide having asequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of Table 1 [SEQ ID NOS:1 or 2] may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In addition to the standard A, G, C, T/U representations for nucleicacid bases, the term “N” may also be used in describing certainpolynucleotides of the invention. “N” means that any of the four DNA orRNA bases may appear at such a designated position in the DNA or RNAsequence, except it is preferred that N is not a base that when taken incombination with adjacent nucleotide positions, when read in the correctreading frame, would have the effect of generating a prematuretermination codon in such reading frame.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, host cells, expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the ribH polynucleotides ofthe invention for use as diagnostic reagents. Detection of ribH in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also“individual(s)”), particularly mammals, and especially humans,particularly those infected or suspected to be infected with an organismcomprising the ribH gene may be detected at the nucleic acid level by avariety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA, cDNA and genomic DNA may also be used in thesame ways. Using amplification, characterization of the species andstrain of prokaryote present in an individual, may be made by ananalysis of the genotype of the prokaryote gene. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the genotype of a reference sequence. Point mutationscan be identified by hybridizing amplified DNA to labeled ribHpolynucleotide sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase digestion or bydifferences in melting temperatures. DNA sequence differences may alsobe detected by alterations in the electrophoretic mobility of the DNAfragments in gels, with or without denaturing agents, or by direct DNAsequencing. See, e.g., Myers et al., Science, 230. 1242 (1985). Sequencechanges at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or a chemicalcleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA,85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA, cDNA or genomic DNA may also be used for the samepurpose, PCR or RT-PCR. As an example, PCR primers complementary to anucleic acid encoding ribH can be used to identify and analyzemutations. Examples of representative primers are shown below in Table2.

TABLE 2 Primers for amplification of ribH polynucleotides SEQ ID NOPRIMER SEQUENCE 3 5′-AAGTTGCAATCGTAGTTAGTCG-3′ 45′-GCTTCGGCACCTTTATTACCA-3′

The invention also includes primers of the formula:

 X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the 5′ end of the molecule, X is hydrogen, and at the 3′ endof the molecule, Y is hydrogen or a metal, R₁ and R₃ is any nucleic acidresidue, m is an integer between 1 and 20 or zero, n is an integerbetween 1 and 20 or zero, and R₂ is a primer sequence of the invention,particularly a primer sequence selected from Table 2. In thepolynucleotide formula above R₂ is oriented so that its 5′ end residueis at the left, bound to R₁, and its 3′ end residue is at the right,bound to R₃. Any stretch of nucleic acid residues denoted by either Rgroup, where m and/or n is greater than 1, may be either a heteropolymeror a homopolymer, preferably a heteropolymer being complementary to aregion of a polynucleotide of Table 1. In a preferred embodiment mand/or n is an integer between 1 and 10.

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5′ and/or the 3′ end. These primers may beused for, among other things, amplifying ribH DNA isolated from a samplederived from an individual. The primers may be used to amplify the geneisolated from an infected individual such that the gene may then besubject to various techniques for elucidation of the DNA sequence. Inthis way, mutations in the DNA sequence may be detected and used todiagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, comprising determining from a sample derived froman individual a increased level of expression of polynucleotide having asequence of Table 1 [SEQ ID NO: 1]. Increased or decreased expression ofribH polynucleotide can be measured using any on of the methods wellknown in the art for the quantation of polynucleotides, such as, forexample, amplification, PCR, RT-PCR, RNase protection, Northern blottingand other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of ribH protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a ribHprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-ribH or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against ribH- polypeptide may be employedto treat infections, particularly bacterial infections.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various formsof liposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS USA 1984:81,5849).

Antagonists and agonists—assays and molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of ribHpolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bacteriocidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagoists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising ribH polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a ribH agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the ribH polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of ribH polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in ribH polynucleotide or polypeptide activity,and binding assays known in the art.

Another example of an assay for ribH antagonists is a competitive assaythat combines ribH and a potential antagonist with ribH-bindingmolecules, recombinant ribH binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. RibH can be labeled, such as byradioactivity or a colorimetric compound, such that the number of ribHmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing ribH-induced activities, thereby preventing the action of ribHby excluding ribH from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of ribH.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock ribH protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial ribH proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat diseases.

Helicobacter pylori (herein H. pylori) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France;http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofribH) found using screens provided by the invention, particularlybroad-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also cure gastric ulcers and gastritis.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with ribH, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Staphylococcus aureus infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector to direct expression of ribH, or afragment or a variant thereof, for expressing ribH, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not. One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise. Such nucleic acid vector may comprise DNA, RNA,a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a ribH or protein coded therefrom,wherein the composition comprises a recombinant ribH or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid ribH or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+ T cells.

A ribH polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain ribHprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, kits and administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

Glossary

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Disease(s)” means and disease caused by or related to infection by abacteria, including disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

“Host cell” is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Commputational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1).387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% “identity” to areference nucleotide sequence of SEQ ID NO: 1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain Selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1]was obtained from a library of clones of chromosomal DNA ofStaphylococcus aureus in E. coli. The sequencing data from two or moreclones containing overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example:

Methods 1 and 2 Below

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E.coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2

RibH Characterization

Characterization of Staphylococcus aureus RibH Gene Expression Geneexpression in vivo

Recently several novel approaches have been described which purport tofollow global gene expression during infection (Chuang, S. et al.,(1993); Mahan, M. J. et al., Science 259:686-688 (1993); Hensel, M. etal., Science 269:400-403 (1995). These new techniques have so far beendemonstrated with gram negative pathogen infections but not withinfections with gram positives presumably because the much slowerdevelopment of global transposon mutagenesis and suitable vectors neededfor these strategies in these organisms, and in the case of that processdescribed by Chuang, S. et al., J. Bacteriol. 175:2026-2036 (1993) thedifficulty of isolating suitable quantities of bacterial RNA free ofmammalian RNA derived from the infected tissue to furnish bacterial RNAlabelled to sufficiently high specific activity.

The present invention employs a novel technology to determine geneexpression in the pathogen at different stages of infection of themammalian host.

Use of the technology of the present invention enables identification ofbacterial genes transcribed during infection, inhibitors of which wouldhave utility in anti-bacterial therapy. Specific inhibitors of such genetranscription or of the subsequent translation of the resultant mRNA orof the function of the corresponding expressed proteins would haveutility in anti-bacterial therapy.

The Determination of Expression During Infection of a Gene fromStaphylococcus aureus WCUH29

Necrotic fatty tissue from a four day groin infection or kidney from aseven day pyelonephritis infection of Staphylococcus aureus WCUH29 inthe mouse is efficiently disrupted and processed in the presence of acidphenol and detergent to provide a mixture of animal and bacterial RNA.By freezing the tissue immediately in liquid nitrogen, and processingthe tissue samples while still frozen, changes in the population ofbacterial mRNA is minimized. The resultant total RNA is free of DNA andprotein (including RNAases and DNAases). The optimal conditions fordisruption and processing to give high yields of bacterial mRNA withtranscripts of long length are followed by reverse transcribing theresulting mRNA to cDNA and amplified with ORF-specific primers for abacterial gene known to be expressed constitutively and at low copynumber in Staphylococcus aureus WCUH29. Aspects of this example II, partb, are modifications of a published protocol (Cheung, et al.; AnalBiochem (1994) 222:511-514).

a) Isolation of Tissue Infected with Staphylococcus aureus WCUH29 fromMurine Models of Infection

i) Isolation of tissue infected with Staphylococcus aureus WCUH29 from amurine thigh lesion model of infection. 10 ml. volumes of sterilenutrient broth (No.2 Oxoid) are seeded with isolated, individualcolonies of Staphylococcus aureus WCUH29 from an agar culture plate. Thecultures are incubated aerobically (static culture) at 37° C. for 16-20hours. 4 week old mice (female, 18 g-22 g, strain MF1) are each infectedby subcutaneous injection of 0.5 ml. of this broth culture ofStaphylococcus aureus WCUH29 (diluted in broth to approximately 10⁸cfu/ml.) into the anterior, right lower quadrant (groin area). Miceshould be monitored regularly during the first 24 hours after infection,then daily until termination of study. Animals with signs of systemicinfection, i.e. lethargy, ruffled appearance, isolation from group,should be monitored closely and if signs progress to moribundancy, theanimal should be culled immediately.

Visible external signs of lesion development will be seen 24-48 h afterinfection. Examination of the abdomen of the animal will show the raisedoutline of the abscess beneath the skin. The localised lesion shouldremain in the right lower quadrant, but may occasionally spread to theleft lower quadrant, and superiorly to the thorax. On occasions, theabscess may rupture through the overlying skin layers. In such cases theaffected animal should be culled immediately and the tissues sampled ifpossible. Failure to cull the animal may result in the necrotic skintissue overlying the abscess being sloughed off, exposing the abdominalmuscle wall.

Approximately 96 hours after infection, animals are killed using carbondioxide asphyxiation. To minimise delay between death and tissueprocessing/storage, mice should be killed individually rather than ingroups. The dead animal is placed onto its back and the fur swabbedliberally with 70% alcohol. An initial incision using scissors is madethrough the skin of the abdominal left lower quadrant, travellingsuperiorly up to, then across the thorax. The incision is completed bycutting inferiorly to the abdominal lower right quadrant. Care should betaken not to penetrate the abdominal wall. Holding the skin flap withforceps, the skin is gently pulled way from the abdomen. The exposedabscess, which covers the peritoneal wall but generally does notpenetrate the muscle sheet completely, is excised, taking care not topuncture the viscera

The abscess/muscle sheet and other infected tissue may require cuttingin sections, prior to flash-freezing in liquid nitrogen, therebyallowing easier storage in plastic collecting vials.

ii) Isolation of tissue infected with Staphylococcus aureus WCUH29 froma murine model of hematogenous pyelonephritis. Overnight cultures of S.aureus WCUH29 are started from single colonies in 5 ml of tryptic soybroth (TSB) and grown at 37 C. with shaking. The cultures are thenwashed twice in sterile phosphate-buffered saline (PBS) and diluted toan A600=0.3. Male CD-1 mice (18-20g) are infected with 0.2 ml of thissuspension by tail vein inoculation using a 30 g needle attached to atuberculin syringe. Each mouse receives approximately 4×107 bacteria inthis fashion. Mice are monitored daily for signs of illness, and usuallywithin 48 hours show signs of lethargy, ruffled fur, sluggishness;animals which appear moribund are euthanized prior to the end of theexperiment.

All animals are euthanized via carbon dioxide overdose seven dayspost-infection. The animal is placed on its back and swabbed withethanol, and then with RNAZap, and instruments are swabbed as well. Theabdominal cavity is opened and the kidneys aseptically removed, cut intofour pieces, and placed in cryovials which are immediately frozen inliquid nitrogen.

b) Isolation of Staphylococcus aureus WCUH29 RNA from Infected TissueSamples

Infected tissue samples, in 2-ml cryo-strorage tubes, are removed fromliquid nitrogen storage for immediate processing of the frozen tissue.In a microbiological safety cabinet the samples are disrupted up toeight at a time. To disrupt the bacteria within the tissue sample,50-100 mg of the tissue is transfered to a FastRNA tube containing asilica/ceramic matrix (BIO101). Immediately, 1 ml of extraction reagents(FastRNA reagents, BIO101) are added to give a sample to reagent volumeratio of approximately 1 to 20. The tubes are shaken in a reciprocatingshaker (FastPrep FP120, BIO101) at a setting of 5.5 to 6 for 20-120 sec.The crude RNA preparation is extracted with chloroform/isoamyl alcohol,and precipitated with DEPC-treated/Isopropanol Precipitation Solution(BIO101). RNA preparations are stored in this isopropanol solution at−80° C. if necessary. The RNA is pelleted (12,000 g for 10 min.), washedwith 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10 min,and resuspended in 0.1 ml of DEPC-treated water.

Quality of the RNA isolated is assessed by the ability to detectbacterial transcripts up to 2 kb in length by RT-PCR (as described insection below). To demonstrate the isolation of bacterial RNA from theinfected tissue, samples of RNA are reverse transcribed, and thepresence of a constitutively expressed gene is detected through the useof quantitative PCR in the presence of a TaqMan probe (as describedbelow).

c) The Removal of DNA from Staphylococcus aureus WCUH29-derived RNA

DNA was removed from 50 microgram samples of RNA by a 30 minutetreatment at 37° C. with 10 units of RNAase-free DNAaseI (GeneHunter) inthe buffer supplied in a final volume of 57 microliters.

The DNAase was inactivated and removed by phenol:chloroform extraction.RNA was precipitated with 5 microliters of 3 M NaOAc and 200 microliters100% EtOH, and pelleted by centrifugation at 12,000 g for 10 minutes.The RNA is pelleted (12,000 g for 10 min.), washed with 75% ethanol (v/vin DEPC-treated water), air-dried for 5-10 min, and resuspended in 10-20microliters of DEPC-treated water. RNA yield is quantitated by OD₂₆₀after 1:1000 dilution of the cleaned RNA sample. RNA is stored at −80°C. if necessary and reverse-transcribed within one week.

d) The Preparation of cDNA from RNA Samples Derived from Infected Tissue

10 microliter samples of DNAase treated RNA are reverse transcribedusing.a SuperScript Preamplification System for First Strand cDNASynthesis kit (Gibco BRL, Life Technologies) according to themanufacturers instructions. 1 nanogram of random hexamers is used toprime each reaction. Controls without the addition of SuperScriptIIreverse transcriptase are also run. Both +/−RT samples are treated withRNaseH before proceeding to the PCR reaction.

e) The Use of PCR to Determine the Quality of Bacterial RNA Derived fromInfected Tissue

Long transcripts, which are expected to be of low copy number within thebacterial cell, such as penicillin-binding protein 2 (PBP2), are reversetranscribed with random primers as described above and amplified by thefollowing PCR method using ORF-specific primers, in order to ascertainthe quality, represented by length amplified, of the mRNA obtainedduring extraction and purification.

PCR reactions are set up on ice in 0.2 ml tubes in a total volume of 50ul by adding the following components [final concentration]: AmpliTaqPCR Buffer II (1X), 1.5 mM MgCL₂, 1 mM dNTPs, 0.5 uM forward primer, 0.5uM reverse primer, and 2 ul reverse-transcribed RNA. PCR reactions arerun on a PE GeneAmp PCR System 9600 with an initial step of 94° C. for 2min, followed by 35 cycles of 94° C. for 30 sec, 42° C. for 30 sec and72° C. for 30 sec, followed by a final extensison at 72° C. for 7 min.

f) The Use of PCR to Determine the Presence of a Bacterial cDNA Species

PCR reactions are set up as described above using 0.5 microM each of theORF specific forward and reverse primers.

PCR product in 20 microliter aliquots are separated by electrophoresisthrough 1 to 1.5% 1×TBE agarose gels or 10% 1×TBE acrylamide gels. PCRproduct is visualized by staining the gel with ethidium bromide. Sizeestimates are made by comparison to a 100 bp DNA Ladder (Gibco BRL, LifeTechnologies). Alternatively, if the PCR products are convenientlylabelled by the use of a labelled PCR primer (e.g. labelled at the 5′endwith a dye) a suitable aliquot of the PCR product is run out on apolyacrylamide sequencing gel and its presence and quantity detectedusing a suitable gel scanning system (e.g. ABI Prism™ 377 Sequencerusing GeneScan™ software as supplied by Perkin Elmer).

RT/PCR controls may include +/− reverse transcriptase reactions, 16 srRNA primers or DNA specific primer pairs designed to produce PCRproducts from non-transcribed Staphylococcus aureus WCUH29 genomicsequences.

To test the efficiency of the primer pairs they are used in DNA PCR withStaphylococcus aureus WCUH29 total DNA. PCR reactions are set up and runas described above using approx. 1 microgram of DNA in place of the cDNAand 35 cycles of PCR.

Primer pairs which fail to give the predicted sized product in eitherDNA PCR or RT/PCR are PCR failures and as such are uninformative. Ofthose which give the correct size product with DNA PCR two classes aredistinguished in RT/PCR: 1.Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR; and 2.Genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stronger signal in the +RT samples than the signal (if at allpresent) in −RT controls.

g) The Use of PCR and Fluorogenic Probes to Determine the Presence of aBacterial cDNA Species

Specific sequence detection occurs by amplification of target sequencesin the PE Applied Biosystems 7700 Sequence Detection System in thepresence of an oligonucleotide probe labeled at the 5′ and 3′ ends witha reporter and quencher fluorescent dye, respectively (FQ probe), whichanneals between the two PCR primers. Only specific product will bedetected when the probe is bound between the primers. As PCRamplification proceeds, the 5′-nuclease activity of Taq polymeraseinitially cleaves the reporter dye from the probe. The signal generatedwhen the reporter dye is physically separated from the quencher dye isdetected by measuring the signal with an attached CCD camera. Eachsignal generated equals one probe cleaved which corresponds toamplification of one target strand.

PCR reactions are set up using the PE Applied Biosystem TaqMan PCR CoreReagent Kit according to the instructions supplied such that eachreaction contains 5 microliters 10X PCR Buffer II, 7 microliters 25 mMMgCl₂, 5 microliters 300 nM forward primer, 5 microliters reverseprimer, 5 microliters specific FQ probe, 1 microliter each 10 mM dATP,10 mM dCTP, 10 mM dGTP and 20 mM dUTP, 13.25 microliters distilledwater, 0.5 microliters AmpErase UNG, and 0.25 microliters AmpliTaq DNApolymerase to give a total volume of 45 microliters.

Amplification proceeds under the following thermal cycling conditions:50° C. hold for 2 minutes, 95° C. hold for 10 minutes, 40 cycles of 95°C. for 15 seconds and 60° C. for 1 minute, followed by a 25° C. holduntil sample is retrieved. Detection occurs real-time. Data is collectedat the end of the reaction.

Based on these analyses it was discovered that the Staphylococcus aureusribH gene was transcribed in vivo.

Riboflavin Biosynthesis Operon

This ORF is part of an operon which encodes genes ribG, ribB, ribA andribH. Gene ribG starts at nucleotide 1 and ends at nucleotide 1029. GeneribB starts at nucleotide 1036 and ends at nucleotide 1668. Gene ribAstarts at nucleotide 1679 and ends at nucleotide 2860. Gene ribH startsat nucleotide 2873 and ends at nucleotide 3337. The operon is as follows(SEQ ID NO: 5)

ATGGATTATGCGATTCAACTTGCAAATATGGTACAAGGTCAAACAGGTGTTAATCCACCCGTTGGCGCTGTTGTAGTTAATGAAGGTAGGATTGTTGGTATTGGTGCACACTTGAGAAAAGGTGACAAGCATGCGGAGGTTCAAGCACTTGATATGGCACAACAAAATGCTGAAGGTGCGACGATTTATATTACGTTAGAGCCATGTAGTCATTTTGGTTCAACACCACCCTGTGTTAACAAAATTATTGATTGTAAGATAGCAAAAGTAGTATACGCAACAAAAGACAATTCGTTAGACACACATGGTGATGAGACGTTACGGGCTCACGGTATTGAGGTTGAATGCGTTGATGATGAACGGGCATCACAATTATACCAAGACTTTTTTAAAGCAAAAGCAAAGCAACTGCCACAAATTACAGTGAAAGTATCTGCAAGTTTAGATGGTAAACAAGCGAATGATAATGGACAAAGTCAATGGATTACTAACAAAGAGGTTAAACAAGATGTCTATAAGTTAAGACATCGACACGACGCAGTGTTAACTGGAAGACGTACAGTTGAATTAGATGATCCACAATATACTACACGTATTCAAGATGGAAAAAACCCTATAAAAGTAATATTGTCTAAGTCTCGGAATATTCATTTTAATCAGCAAATTTATCAAGATGAATCAACACCAATTTGGATATATACTGAAAATCCAAATTTAACAAGCAATCAAACACATATTGAAATTATTTACTTGAAGTCTTGTGATTTAACAACAATTCTTCACAATTTATATAAAAGAGGAGTTGGAACTTTGCTAGTCGAGGCAGGTCCAACCACTACTTCAGAATTCTCCATCTATTATATAGATGAATTTATTCTCTATTATGCCCCGAAATTAATTGGCGGATCTGGAAATTATCAATTTTATCAAACAAATGATGTGATTGAGATACCAGATGCGAACCAATTTGAAATTGTTCATTCCGAGTTATTAAATCAAAATGTTAAATTAACTTTACGAAAGAAGTGATGATGCATGTTTACTGGCATCGTTGAAGAAATAGGTGTCGTTAAAAGTGTTCAAATTCGTCAATCAGTCAGGACGATTGAAATCGAAGCACATAAGATTACGGCAGATATGCATATTGGTGATTCCATCAGTGTTAATGGTGCATGTTTAACAGTGATTGATTTTGATCAGACATCTTTTACTGTTCAAGTAATTAAAAGCACTGAAAATAAAACCTATTTAGCAGATGTTAAGCGACAATCAGAAGTAAATTTAGAGCGTGCCATGAGTGGTAACGGTAGGTTTGGTGGACATTTTGTCCTCGGTCATGTAGATGAACTAGGAACAGTTTCAAAAATAAATGAAACAGCCAATGCCAAAATTATTACGATTCAATGTAGCCAACATATTAATAATCAGTTAGTTAAGCAAGGGTCTATTACTGTGGATGGTGTAAGTCTAACGGTATTTGATAAGCATGATAACAGTTTTGACATTCATCTTATTCCAGAAACGAGGCGTTCAACGATTTTATCATCCAAAAAATTAGGAGATAAAGTACATTTAGAAACAGACGTTTTGTTTAAATATGTTGAAAATATTTTAAATAAAGATAAAGACCAATTATCTGTAGATAAATTAAGAGCATTTGGTTTTTAGGAGGGGTAGCATGCAATTCGATAATATTGACAGTGCTTTAATGGCTTTAAAAAATGGAGAAACAATTATTGTAGTAGATGATGAGAATCGTGAAAATGAAGGTGATTTAGTAGCGGTTACTGAATGGATGAACGATAATACCATTAATTTTATGGCGAAAGAAGCAAGGGGATTAATATGCGCACCAGTGTCTAAAGATATTGCACAACGTTTGGATTTGGTACAAATGGTTGATGATAACTCCGACATCTTTGGTACGCAATTTACAGTGAGTATTGATCATGTAGATACAACAACAGGAATTAGTGCTTATGAACGTACATTGACTGCCAAAAAGCTCATTGATCCTAGTAGTGAAGCTAAAGATTTTAATCGTCCTGGTCATTTATTTCCATTAGTAGCACAAGATAAAGGCGTATTAGCTAGAAATGGACACACAGAAGCGGCTGTTGATTTAGCTAAACTTACTGGTGCCAAGCCCGCTGGTGTCATTTGTGAGATTATGAATGATGACGGCACGATGGCGAAAGGACAAGATTTACAAAATTTTAAAGAAAAACATCAATTAAAGATGATTACGATTGATGATTTAATTGAATATCGTAAAAATTAGAACCAGAAATTGAATTTAAGGCAAAAGTGAAAATGCCTACAGATTTCGGAACATTTGATATGTATGGTTTTAAAGCGACATACACAGATGAAGAGATAGTTGTACTGACAAAAGGTGCAATTCGACAACATGAAAATGTACGCTTACATTCTGCGTGCCTTACAGGCGATATTTTCCATAGTCAACGTTGTGATTGTGGTGCTCAACTTGAATCGTCTATGAAGTATATCAATGAACATGGTGGCATGATTATTTATCTACCTCAAGAAGGTCGTGGCATAGGATTGTTAAACAAATTACGCGCATATGAATTAATTGAGCAAGGATATGATACAGTAACTGCAAATTTAGCATTAGGTTTTGATGAAGATTTACGAGATTATCATATTGCTGCACAGATTTTAAAATATTTTAACATCGAACATATCAATTTATTAAGTAATAATCCAAGTAAATTTGAGGGATTAAAACAATATGGCATTGATATTGCAGAAAGAATTGAAGTTATCGTACCAGAAACGGTACATAATCATGATTATATGGTAACGAAAAAAATAAAAATGGGTCATTTAATATAGGAGGACTTTAACATGAATTTTGAAGGTAAATTAATTGGAAAAGATTTGAAAGTTGCAATCGTAGTTAGTCGATTTAATGATTTTATCACTGGAAGATTACTTGAAGGTGCAAAAGATACTTTGATTCGACATGATGTTAATGAAGACAATATTGATGTAGCATTTGTTCCTGGTGCGTTTGAAATTCCTTTAGTAGCTAAAAAATTAGCCTCATCAGGAAATTATGATGCAATAATTACATTAGGATGCGTAATTCGCGGTGCTACGTCTCATTATGATTATGTTTGTAATGAAGTGCGAAAGGTGTTTCTAAAGTAAATGATCAAACTAATGTACCAGTCATATTTGGTATTTTAACGACTGAAAGTATTGAACAAGCTGTGGAAAGAGCAGGTACGAAAGCTGGTAATAAAGGTGCCGAAGCAGCAGTAAGTGCAATTGAAATGGCTAATTTATTAAAATCTATAAAAGCATAG

5 465 base pairs nucleic acid double linear not provided 1 ATGAATTTTGAAGGTAAATT AATTGGAAAA GATTTGAAAG TTGCAATCGT AGTTAGTCGA 60 TTTAATGATTTTATCACTGG AAGATTACTT GAAGGTGCAA AAGATACTTT GATTCGACAT 120 GATGTTAATGAAGACAATAT TGATGTAGCA TTTGTTCCTG GTGCGTTTGA AATTCCTTTA 180 GTAGCTAAAAAATTAGCCTC ATCAGGAAAT TATGATGCAA TAATTACATT AGGATGCGTA 240 ATTCGCGGTGCTACGTCTCA TTATGATTAT GTTTGTAATG AAGTCGCGAA AGGTGTTTCT 300 AAAGTAAATGATCAAACTAA TGTACCAGTC ATATTTGGTA TTTTAACGAC TGAAAGTATT 360 GAACAAGCTGTGGAAAGAGC AGGTACGAAA GCTGGTAATA AAGGTGCCGA AGCAGCAGTA 420 AGTGCAATTGAAATGGCTAA TTTATTAAAA TCTATAAAAG CATAG 465 154 amino acids amino acidsingle linear not provided 2 Met Asn Phe Glu Gly Lys Leu Ile Gly Lys AspLeu Lys Val Ala Ile 1 5 10 15 Val Val Ser Arg Phe Asn Asp Phe Ile ThrGly Arg Leu Leu Glu Gly 20 25 30 Ala Lys Asp Thr Leu Ile Arg His Asp ValAsn Glu Asp Asn Ile Asp 35 40 45 Val Ala Phe Val Pro Gly Ala Phe Glu IlePro Leu Val Ala Lys Lys 50 55 60 Leu Ala Ser Ser Gly Asn Tyr Asp Ala IleIle Thr Leu Gly Cys Val 65 70 75 80 Ile Arg Gly Ala Thr Ser His Tyr AspTyr Val Cys Asn Glu Val Ala 85 90 95 Lys Gly Val Ser Lys Val Asn Asp GlnThr Asn Val Pro Val Ile Phe 100 105 110 Gly Ile Leu Thr Thr Glu Ser IleGlu Gln Ala Val Glu Arg Ala Gly 115 120 125 Thr Lys Ala Gly Asn Lys GlyAla Glu Ala Ala Val Ser Ala Ile Glu 130 135 140 Met Ala Asn Leu Leu LysSer Ile Lys Ala 145 150 22 base pairs nucleic acid single linear notprovided 3 AAGTTGCAAT CGTAGTTAGT CG 22 21 base pairs nucleic acid singlelinear not provided 4 GCTTCGGCAC CTTTATTACC A 21 3336 base pairs nucleicacid double linear not provided 5 ATGGATTATG CGATTCAACT TGCAAATATGGTACAAGGTC AAACAGGTGT TAATCCACCC 60 GTTGGCGCTG TTGTAGTTAA TGAAGGTAGGATTGTTGGTA TTGGTGCACA CTTGAGAAAA 120 GGTGACAAGC ATGCGGAGGT TCAAGCACTTGATATGGCAC AACAAAATGC TGAAGGTGCG 180 ACGATTTATA TTACGTTAGA GCCATGTAGTCATTTTGGTT CAACACCACC CTGTGTTAAC 240 AAAATTATTG ATTGTAAGAT AGCAAAAGTAGTATACGCAA CAAAAGACAA TTCGTTAGAC 300 ACACATGGTG ATGAGACGTT ACGGGCTCACGGTATTGAGG TTGAATGCGT TGATGATGAA 360 CGGGCATCAC AATTATACCA AGACTTTTTTAAAGCAAAAG CAAAGCAACT GCCACAAATT 420 ACAGTGAAAG TATCTGCAAG TTTAGATGGTAAACAAGCGA ATGATAATGG ACAAAGTCAA 480 TGGATTACTA ACAAAGAGGT TAAACAAGATGTCTATAAGT TAAGACATCG ACACGACGCA 540 GTGTTAACTG GAAGACGTAC AGTTGAATTAGATGATCCAC AATATACTAC ACGTATTCAA 600 GATGGAAAAA ACCCTATAAA AGTAATATTGTCTAAGTCTG GGAATATTCA TTTTAATCAG 660 CAAATTTATC AAGATGAATC AACACCAATTTGGATATATA CTGAAAATCC AAATTTAACA 720 AGCAATCAAA CACATATTGA AATTATTTACTTGAAGTCTT GTGATTTAAC AACAATTCTT 780 CACAATTTAT ATAAAAGAGG AGTTGGAACTTTGCTAGTCG AGGCAGGTCC AACCACTACT 840 TCAGAATTCT CCATCTATTA TATAGATGAATTTATTCTCT ATTATGCCCC GAAATTAATT 900 GGCGGATCTG GAAATTATCA ATTTTATCAAACAAATGATG TGATTGAGAT ACCAGATGCG 960 AACCAATTTG AAATTGTTCA TTCCGAGTTATTAAATCAAA ATGTTAAATT AACTTTACGA 1020 AAGAAGTGAT GATGCATGTT TACTGGCATCGTTGAAGAAA TAGGTGTCGT TAAAAGTGTT 1080 CAAATTCGTC AATCAGTCAG GACGATTGAAATCGAAGCAC ATAAGATTAC GGCAGATATG 1140 CATATTGGTG ATTCCATCAG TGTTAATGGTGCATGTTTAA CAGTGATTGA TTTTGATCAG 1200 ACATCTTTTA CTGTTCAAGT AATTAAAAGCACTGAAAATA AAACCTATTT AGCAGATGTT 1260 AAGCGACAAT CAGAAGTAAA TTTAGAGCGTGCCATGAGTG GTAACGGTAG GTTTGGTGGA 1320 CATTTTGTCC TCGGTCATGT AGATGAACTAGGAACAGTTT CAAAAATAAA TGAAACAGCC 1380 AATGCCAAAA TTATTACGAT TCAATGTAGCCAACATATTA ATAATCAGTT AGTTAAGCAA 1440 GGGTCTATTA CTGTGGATGG TGTAAGTCTAACGGTATTTG ATAAGCATGA TAACAGTTTT 1500 GACATTCATC TTATTCCAGA AACGAGGCGTTCAACGATTT TATCATCCAA AAAATTAGGA 1560 GATAAAGTAC ATTTAGAAAC AGACGTTTTGTTTAAATATG TTGAAAATAT TTTAAATAAA 1620 GATAAAGACC AATTATCTGT AGATAAATTAAGAGCATTTG GTTTTTAGGA GGGGTAGCAT 1680 GCAATTCGAT AATATTGACA GTGCTTTAATGGCTTTAAAA AATGGAGAAA CAATTATTGT 1740 AGTAGATGAT GAGAATCGTG AAAATGAAGGTGATTTAGTA GCGGTTACTG AATGGATGAA 1800 CGATAATACC ATTAATTTTA TGGCGAAAGAAGCAAGGGGA TTAATATGCG CACCAGTGTG 1860 TAAAGATATT GCACAACGTT TGGATTTGGTACAAATGGTT GATGATAACT CCGACATCTT 1920 TGGTACGCAA TTTACAGTGA GTATTGATCATGTAGATACA ACAACAGGAA TTAGTGCTTA 1980 TGAACGTACA TTGACTGCCA AAAAGCTCATTGATCCTAGT AGTGAAGCTA AAGATTTTAA 2040 TCGTCCTGGT CATTTATTTC CATTAGTAGCACAAGATAAA GGCGTATTAG CTAGAAATGG 2100 ACACACAGAA GCGGCTGTTG ATTTAGCTAAACTTACTGGT GCCAAGCCCG CTGGTGTCAT 2160 TTGTGAGATT ATGAATGATG ACGGCACGATGGCGAAAGGA CAAGATTTAC AAAATTTTAA 2220 AGAAAAACAT CAATTAAAGA TGATTACGATTGATGATTTA ATTGAATATC GTAAAAAATT 2280 AGAACCAGAA ATTGAATTTA AGGCAAAAGTGAAAATGCCT ACAGATTTCG GAACATTTGA 2340 TATGTATGGT TTTAAAGCGA CATACACAGATGAAGAGATA GTTGTACTGA CAAAAGGTGC 2400 AATTCGACAA CATGAAAATG TACGCTTACATTCTGCGTGC CTTACAGGCG ATATTTTCCA 2460 TAGTCAACGT TGTGATTGTG GTGCTCAACTTGAATCGTCT ATGAAGTATA TCAATGAACA 2520 TGGTGGCATG ATTATTTATC TACCTCAAGAAGGTCGTGGC ATAGGATTGT TAAACAAATT 2580 ACGCGCATAT GAATTAATTG AGCAAGGATATGATACAGTA ACTGCAAATT TAGCATTAGG 2640 TTTTGATGAA GATTTACGAG ATTATCATATTGCTGCACAG ATTTTAAAAT ATTTTAACAT 2700 CGAACATATC AATTTATTAA GTAATAATCCAAGTAAATTT GAGGGATTAA AACAATATGG 2760 CATTGATATT GCAGAAAGAA TTGAAGTTATCGTACCAGAA ACGGTACATA ATCATGATTA 2820 TATGGTAACG AAAAAAATAA AAATGGGTCATTTAATATAG GAGGACTTTA ACATGAATTT 2880 TGAAGGTAAA TTAATTGGAA AAGATTTGAAAGTTGCAATC GTAGTTAGTC GATTTAATGA 2940 TTTTATCACT GGAAGATTAC TTGAAGGTGCAAAAGATACT TTGATTCGAC ATGATGTTAA 3000 TGAAGACAAT ATTGATGTAG CATTTGTTCCTGGTGCGTTT GAAATTCCTT TAGTAGCTAA 3060 AAAATTAGCC TCATCAGGAA ATTATGATGCAATAATTACA TTAGGATGCG TAATTCGCGG 3120 TGCTACGTCT CATTATGATT ATGTTTGTAATGAAGTGCGA AAGGTGTTTC TAAAGTAAAT 3180 GATCAAACTA ATGTACCAGT CATATTTGGTATTTTAACGA CTGAAAGTAT TGAACAAGCT 3240 GTGGAAAGAG CAGGTACGAA AGCTGGTAATAAAGGTGCCG AAGCAGCAGT AAGTGCAATT 3300 GAAATGGCTA ATTTATTAAA ATCTATAAAAGCATAG 3336

What is claimed is:
 1. An isolated polypeptide comprising SEQ ID NO:2,wherein the isolated polypeptide exhibits lumazine synthase activity. 2.The isolated polypeptide of claim 1, wherein the isolated polypeptidecomprises a heterologous amino acid sequence fused to SEQ ID NO:2. 3.The isolated polypeptide of claim 1, wherein the isolated polypeptideconsists of SEQ ID NO:2.
 4. An isolated polypeptide comprising afragment of SEQ ID NO:2 comprising at least 50 consecutive amino acidsof SEQ ID NO:2; wherein said fragment exhibits riboflavin lumazinesynthase activity.
 5. The isolated polypeptide of claim 4, wherein theisolated polypeptide comprises a heterologous amino acid sequence fusedto the at least 50 consecutive amino acids of SEQ ID NO:2.
 6. Anisolated polypeptide comprising a fragment of SEQ ID NO:2 comprising atleast 30 consecutive amino acids of SEQ ID NO:2; wherein said fragmentexhibits riboflavin lumazine synthase activity.
 7. The isolatedpolypeptide of claim 6, wherein the isolated polypeptide comprises aheterologous amino acid sequence fused to the at least 30 consecutiveamino acids of SEQ ID NO:2.